Yamakura F, Taka H, Fujimura T, Murayama K
Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba 270-16, Japan.
J Biol Chem. 1998 Jun 5;273(23):14085-9. doi: 10.1074/jbc.273.23.14085.
Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O-2 gateway in Mn-SOD.
最近有研究表明过氧亚硝酸盐与许多酶的失活有关。然而，关于失活反应的结构基础却鲜有报道。本研究提出，特定酪氨酸残基的硝化作用是过氧亚硝酸盐导致重组人线粒体锰超氧化物歧化酶（Mn-SOD）失活的原因。对过氧亚硝酸盐失活的Mn-SOD进行质谱分析，结果显示由于一个硝基取代到酪氨酸残基上，分子量增加。在用葡萄球菌丝氨酸蛋白酶和赖氨酰内肽酶对样品进行连续消化后，分离出了在反相高效液相色谱上来自天然和过氧亚硝酸盐失活的Mn-SOD样品之间具有不同洗脱位置的单个肽段，并对其进行了氨基酸序列和分子量分析。我们发现该酶的酪氨酸34被过氧亚硝酸盐专一性硝化为3-硝基酪氨酸。该残基位于锰附近且处于Mn-SOD的底物O₂通道中。
